Method for non-homologous transformation of Yarrowia lipolytica

ABSTRACT

The invention concerns the integration of a gene of interest into the genome of a Yarrowia strain devoid of zeta sequences, by transforming said strain using a vector bearing zeta sequences.

The invention relates to tools for expressing heterologous genes inYarrowia lipolytica.

The yeast Y. lipolytica is being increasingly used as a host forexpressing genes of interest; in this context, “integrating” vectors,which allow the insertion of a segment of DNA bearing the gene ofinterest into the chromosomal DNA, are in particular used. For example,Application EP 138 508 discloses the transformation of Yarrowialipolytica using vectors capable of integrating into the chromosomal DNAby recombination of Y. lipolytica sequences borne by said vectors, withthe homologous sequences present on the chromosomal DNA of the hostcell.

Among the Yarrowia lipolytica sequences used for constructingintegrating vectors, mention will be made in particular of the sequencestermed: “zeta sequences”, which correspond to the LTRs (long terminalrepeats) of the Ylt1 retrotransposon of Yarrowia lipolytica; thesesequences have been described by SCHMID-BERGER et al. [J. Bacteriol.,2477-2482 (1994)], who indicate that they are present at a high copynumber (approximately 35 copies of the complete retrotransposon andapproximately 30 copies of the isolated zeta sequence) in thechromosomal DNA of certain strains of Yarrowia lipolytica. When a vectorcontaining an insert flanked by zeta sequences is used to transform oneof these strains of Yarrowia, the insert DNA integrates by homologousrecombination with zeta sequences of the chromosomal DNA. In this way,transformed Yarrowia cells containing several copies of a heterologoussequence, integrated in tandem at chromosomal zeta sites, are obtained.

The inventors have now noted that, surprisingly, when vectors bearinginserts flanked by zeta sequences are used to transform Yarrowia cellslacking these sequences, the insert DNA integrates, however, into thechromosomal DNA, in the form of several copies dispersed in the genome.

A subject of the present invention is a method for integrating a gene ofinterest into the genome of a strain of Yarrowia, using a recombinantvector bearing an insert flanked by zeta sequences and comprising saidgene of interest, which method is characterized in that said recombinantvector is used to transform a strain of Yarrowia, the genome of whichlacks zeta sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: construction of the JMP3 vector.

FIG. 2: the plasmids JMP6 and JMP10.

FIG. 3: results obtained for the PO1d transformants.

FIG. 4: results obtained for the E150 transformants.

Strains of Yarrowia lipolytica lacking zeta sequences, which can be usedfor implementing the method in accordance with the invention, are, forexample, the strains derived from the strain W29 (ATCC 20460 or CLIB 89,MatA), such as W29ura3-302 (CLIB 141, MatA Ura3-302), PO1a (CLIB 140,MatA Ura3-302, Leu2-270) and PO1d (CLIB 139, MatA Ura3-302, Leu2-270,xpr2-322), which are described by BARTH and GAILLARDIN [The dimorphicfungus Yarrowia lipolytica. In: Non conventional yeasts in biotechnology(Wolf K. Ed.). Springer-Verlag, Berlin, pp. 313-388 (1996)], and whichcan be obtained from the CLIB (Collection de Levures d'intérêtBiotechnologique [Collection of yeasts of biotechnological interest],INRA, Centre de Grignon, BP01, 78850 Thiberval-Grignon). Other strainswhich may be used can easily be selected based on the absence of signalsof hybridization with nucleic acid probes derived from the zetasequences.

According to a preferred embodiment of the method in accordance with theinvention, the insert comprising the gene of interest also comprisessequences allowing the control of the expression of said gene and/or atleast one marker for selection of the transformants.

The selection marker consists of a gene whose expression allows theselection of the transformants. It can be, for example, a gene forresistance to an antibiotic, or a mutant defective for a gene requiredfor yeast growth, such as URA3, LEU2, etc. Advantageously, a selectionmarker will be chosen which needs to be present in several copies inorder to be functional, thereby making it possible to select thetransformants which have integrated several copies of the gene ofinterest. For example, use may be made, as a selection marker, of adefective URA3 marker, which is derived from the URA3 gene of Y.lipolytica, which allows complementation of auxotrophy for uracil, suchas the URA3d markers described by LE DALL et al. [Curr. Genet., 26,38-44 (1994)].

The sequences for controlling the expression are, in particular,promoter and terminator sequences which are active in Yarrowia. Use maybe made of an inducible or constitutive promoter.

Very advantageously, use may also be made, as control sequences, of thepromoter of the acyl CoA oxidase gene ACO2 of Yarrowia lipolytica [LECLAINCHE, doctoral thesis from the Institut National Agronomique ParisGrignon [National Agronomic Institute Paris Grignon], defended on Jul.2, 1997], or the promoter and/or terminator of the acid-resistantextracellular lipase gene LIP2 of Yarrowia lipolytica, disclosed in theFrench application under the name of LABORATOIRES MAYLOY-SPINDLER,entitled: “CLONAGE ET EXPRESSION DE LIPASES EXTRACELLULAIRESACIDORESISTANTES DE LEVURES” [Cloning and expression of acid-resistantextracellular lipases of yeasts], filed on the same day as the presentapplication.

The ACO2 and LIP2 promoters can both be induced with triglycerides andfatty acids.

Other selection markers and control sequences which can be used forimplementing the present invention are, for example, those cited byBARTH and GAILLARDIN (abovementioned publication).

When the product of the gene of interest is intended to be secreted bythe host cell, said insert also comprises signals for controlling thesecretion of said product. For this purpose, use may be made of signalsequences which are functional in Yarrowia lipolytica, for example allor part of the prepro sequence of the LIP2 gene disclosed in theabovementioned French application under the name of LABORATOIRESMAYOLY-SPINDLER.

A subject of the present invention is also transformed Yarrowia cellswhich can be obtained using the method in accordance with the invention.Advantageously, these transformed cells comprise at least two copies ofthe insert flanked by zeta sequences, comprising the gene of interest,integrated into their genome in a dispersed manner.

The present invention will be more fully understood with the aid of thefurther description which will follow, which refers to nonlimitingexamples for obtaining transformed strains of Yarrowia lipolytica inaccordance with the invention.

EXAMPLE 1

Construction of Vectors Comprising a ZETA Sequence

These vectors are obtained from the vector pINA1067, derived from avector of type pINA970 described by BARTH and GAILLARDIN (abovementionedpublication, FIG. 6), by excision of the portion of sequence between theXPR2 promoter and terminator, which was replaced with a linkercontaining the SrfI and BglII restriction sites. pINA1067 bears thedefective URA3 marker ura3d4 described by LE DALL et al. (abovementionedpublication), and also a tetracycline resistance marker.

The plasmid pHSS6 bearing an origin of replication for E. coli and akanamycin resistance gene is linearized by restriction with NotI, andintroduced into the NotI site of pINA1067. The ligation mixture is usedto transform E. coli. The transformants which have integrated theplasmid are selected on tetracycline (6 mg/l) and kanamycin (40 mg/l).

The resulting vector, termed JMP1, is cleaved with HindIII and EcoRI,which eliminates the fragment bearing the XPR2 promoter and terminatorsequences and the tetracycline resistance marker; this fragment isreplaced with a polylinker which contains the HindIII, ClaI, MluI, HpaI,BamHI and EcoRI restriction sites.

The resulting vector is termed JMP3. A diagram of the various steps inthe construction of JMP3 is given in FIG. 1.

The self-cloning vectors JMP3 and JMP5 integrate into the genome aftercleavage with the NotI restriction enzyme. This restriction allows theelimination of the bacterial portion (origin of replication of E. coliand of the KanR resistance marker).

EXAMPLE 2

Construction of Expression Vectors

Expression vectors comprising a sequence encoding the acid-resistantextracellular lipase of Yarrowia lipolytica, disclosed in theabovementioned French application under the name of LABORATOIRESMAYOLY-SPINDLER, under the control of the ACO2 promoter (vector JMP6) orof its own promoter (vector JMP10), were constructed.

Vector JMP6

The promoter of the acyl CoA oxidase gene ACO2 was amplified by PCRusing the total genomic DNA of Y. lipolytica, with the oligonucleotidesAco2P1 and Aco2P2, which contain a ClaI site and a HindIII siterespectively.

Aco2P1:

5′ GCG ATCGAT CATACTGTTACACTGCTCCG 3′ (SEQ ID NO: 1)

Aco2P2:

5′ GTGGGATCCGAAAGCTTCATGGCGTCGTTGCTTGTGTGATTTTTGAGG 3′ (SEQ ID NO: 2)

The 2168 bp PCR fragment was cloned into the EcoRV site of the vectorBLUESCRIPT KS+. The resulting plasmid is called KS-ACO2prom. This vectorwas digested with ClaI and partially with HindIII so as to obtain a2155bp fragment containing the complete ACO2 promoter.

In addition, a 1134bp HindIII-EcoRI fragment containing the sequenceencoding the prolipase LIP2, and also the transcription terminator, wasisolated from the plasmid termed pKS+-LIP2W29a. The two purifiedfragments were fused and inserted into the ClaI-EcoRI sites of thevector JMP3. The resulting plasmid is called JMP6.

Vector JMP10

A 2030 bp NdeI*-HindIII fragment containing the promoter of the lipasegene of Y. lipolytica was isolated from the plasmid termedpKS+-LIP2prom. For this, the plasmid pKS+-LIP2prom was digested withNdeI, and then treated with T4 DNA polymerase so as to make the NdeIsite blunt (NdeI*). After inactivation of the NdeI enzyme and of thepolymerase, the DNA was cleaved with HindIII. The 2030 bp NdeI*-HindIIIfragment was ligated with the 1134 bp HindIII-EcoRI fragment bearing thelipase gene and its terminator, and the ligation product was insertedinto the HpaI-EcoRI sites of the vector JMP3. The resulting plasmid iscalled JMP10.

FIG. 2 represents the plasmids JMP6 and JMP10.

EXAMPLE 3

Production of Y. lipolytica Transformants

The following strains of Y. lipolytica, having a deletion of the URA3gene, were used:

PO1d: (CLIB 139, MatA Ura3-302, Leu2-270, xpr2-322), lacking zetasequences;

E150: (CLIB 122, MatA Ura3-302, Leu2-270, xpr2-322, his-1) havingseveral copies of zeta sequences.

These two strains, described by BARTH and GAILLARDIN (abovementionedpublication), are available from the CLIB.

The plasmids JMP6 and JMP10, linearized beforehand with NotI, whichmakes it possible to eliminate the sequences originating from theplasmid pHSS6, are introduced into the Yarrowia cells by transformationwith lithium acetate, according to the protocol described by BARTH andGAILLARDIN.

20 to 50 transformants/μg of DNA are usually obtained with the strainPO1d, and 100 to 200 transformants/μg of DNA with the strain E150.

The various transformants will be referred to hereinafter according tothe following nomenclature: strain name—plasmid number—transformantnumber. For example; PO1d-6-15=strain PO1d, plasmid JMP6, transformantnumber 15.

The structure of the transformants was studied by Southern transfer. Thetotal genomic DNA of the strain of origin (T) and of the varioustransformants is digested with HindIII in the case of PO1d, and withBamHI (for the JMP6 transformants) or EcoRI (for the JMP10transformants) in the case of E150.

The probes used are derived from the zeta sequences, from the sequenceof the ACO2 promoter and from the sequence encoding the lipase LIP2.

The results obtained are illustrated in FIG. 3 for the PO1dtransformants, and in FIG. 4 for the E150 transformants.

PO1d Transformants (JMP6)

FIG. 3A represents the results observed with the lipase probe; FIG. 3Brepresents the results observed with the ZETA probe; FIG. 3C representsthe results observed with the ACO2 probe.

In the strain PO1d (line T), a 1.4 kb band is observed with the lipaseprobe, no band is observed with the zeta probe (absence of a zeta sitein this strain) and a 1.1 kb band is observed with the ACO2 probe. Inthe case of the transformants, with the lipase and zeta probes, manybands of varying size are observed, corresponding to the number ofcopies and showing that they are dispersed in the genome.

In the case of the transformant strains in accordance with theinvention, a non-homologous, multicopy and dispersed integration istherefore observed.

E150 Transformants (JMP6 and JMP10)

FIG. 4A represents the results observed with the lipase probe+the ACO2probe; FIG. 4B represents the results observed with the ZETA probe.

In FIG. 4A, in the transformants, amplification of the vector sequencesis observed, revealed by the intensity of the 2.9 kb BamHI band (ACO2promoter+lipase gene) or of the 1.6 kb EcoRI band (lipase gene),compared with that of the 2.6 kb genomic BamHI band which corresponds tothe genomic ACO2 promoter, or that of the approximately 6 kb genomicEcoRI band which corresponds to the lipase gene.

In FIG. 4B, in the strain E150 and the transformants, several zeta sitesare observed, revealed by many bands in the EcoRI restriction profile.The tandem integration is revealed by the intensity of the 2.5 kb BamHIband and the 3.7 kb EcoRI band, which correspond to the fragmentsexpected if the sequences of the vectors are integrated in tandem.

In transformant 6, the disappearance of a zeta band is also noted,indicating the integration into this locus.

In the case of the strain E150 transformants, tandem integration byhomologous integration at a zeta site is therefore observed.

Lipase Secretion by the Transformed Strains

Lipase production by the transformed strains was tested on variousmedia. Both in the case of the strains in accordance with the inventionderived from PO1d and in that of the comparison strains derived fromE150, secretion at least ten to fifteen times greater than that of thenontransformed strains is observed.

2 1 29 DNA Artificial Sequence Synthetic DNA 1 gcgatcgatc atactgttacactgctccg 29 2 48 DNA Artificial Sequence Synthetic DNA 2 gtgggatccgaaagcttcat ggcgtcgttg cttgtgtgat ttttgagg 48

What is claimed is:
 1. Method for integrating a gene of interest intothe genome of a strain of Yarrowia, using a recombinant vector bearingan insert flanked by zeta sequences and comprising said gene ofinterest, characterized in that said recombinant vector is used totransform a strain of Yarrowia, the genome of which lacks zetasequences.
 2. Method according to claim 1, characterized in that theinsert comprising the gene of interest also comprises sequences allowingthe control of the expression of said gene and/or at least one markerfor selection of the transformants.
 3. Method according to claim 2,characterized in that said selection marker is a defective URA3 marker.4. Method according to claim 2, characterized in that at least one ofthe sequences allowing the control of the expression of the gene ofinterest is chosen from: the promoter of the acyl CoA oxidase gene ACO2of Yarrowia lipolytica, the promoter of the acid-resistant extracellularlipase gene of Yarrowia lipolytica and the terminator of theacid-resistant extracellular lipase gene of Yarrowia lipolytica. 5.Method according to claim 2, characterized in that said expressioncassette also comprises a sequence encoding signals for controlling thesecretion of the product of said gene of interest.
 6. Method accordingto claim 5, characterized in that said control signals comprise all orpart of the prepro sequence of the acid-resistant extracellular lipasegene of Yarrowia lipolytica.
 7. Transformed Yarrowia cell which can beobtained using a method according to claim
 1. 8. Transformed cellaccording to claim 7, characterized in that it comprises at least 2copies of the sequences of said vector, integrated into its genome in adispersed manner.
 9. The method of claim 1, wherein the transformedstrain has at least two copies of said vector, integrated into itsgenome in a dispersed manner.
 10. The method of claim 1, wherein theYarrowia strain is Yarrowia lipolytica.
 11. The method of claim 10,wherein the Yarrowia strain is W29ura3-302, PO1a, or PO1d.
 12. Thetransformed Yarrowia cell of claim 7, which is Yarrowia lipolytica. 13.The transformed Yarrowia cell of claim 12, wherein the Yarrowia strainW29ura3-302, PO1a, or PO1d is transformed.
 14. Transformed Yarrowia cellwhich is obtained by the method of claim
 2. 15. Transformed Yarrowiacell which is obtained by the method of claim
 3. 16. TransformedYarrowia cell which is obtained by the method of claim
 4. 17.Transformed Yarrowia cell which is obtained by the method of claim 5.18. Transformed Yarrowia cell which is obtained by the method of claim6.
 19. Transformed Yarrowia cell which is obtained by the method ofclaim
 9. 20. Transformed Yarrowia cell which is obtained by the methodof claim
 10. 21. Transformed Yarrowia cell which is obtained by themethod of claim 11.